Several clinically important classes of antibiotics strongly induce the SOS response in bacteria. SOS defective bacteria are significantly more sensitive to quinolone, fluoroquinolone, and 2-lactam antibiotics than are wild type bacteria. Induction of the SOS response increases the evolution of antibiotic resistance and the horizontal transfer of virulence factors. Therefore, the SOS induction pathway is an important target for discovery of drugs that act as potentiators of clinically important antibiotics and reduce the evolution of antibiotic resistance. The overall goal of this STTR proposal is to discover and develop drugs that are potentiators of clinically relevant fluoroquinolones by specifically inhibiting induction of the SOS response. This approach represents an innovation in the way bacterial infections will be treated. These innovative drugs will increase the efficacy of existing clinically relevant antibiotics at low doses, prevent evolution of resistance and transmission of virulence factors. In Phase I of this STTR, we will identify inhibitors of SOS induction using a cell-based reporter assay in a high-throughput screen (HTS) of a diverse library of drug-like small molecules. We will evaluate these inhibitors for potency, specificity, and spectrum of activity using a series of secondary assays. The activity of the SOS inhibitors will be optimized in iterative rounds of medicinal chemistry and evaluation. Inhibitors that meet the criteria specified for secondary assays will be designated Validated Hits. Mechanism of Action (MOA) studies will be carried out for promising Validated Hits. In Phase II, Validated Hits with a confirmed MOA will be developed into Lead Compounds by optimizing their activity and specificity using rational drug design. The Specific Aims are as follows. Aim 1- Develop a cell-based reporter HTS for inhibitors of SOS induction. Aim 2-Screen a diverse compound library, identify, and confirm inhibitors of SOS induction. Aim 3-Validate confirmed SOS inhibitors using secondary assays and identify a validated Hit series. Aim 4-Determine mechanism of action of Validated Hits/Identify target. PUBLIC HEALTH RELEVANCE: Bacteria respond to environmental insults by producing proteins that increase their survival. Several antibiotics that are currently used to treat bacterial infections, such as ampicillin, amoxacillin, and ciprofloxacin, activate the production of a set of proteins collectively referred to as the SOS response. This important stress response plays a critical role in repairing DNA damage and increasing tolerance to DNA damaging agents. Bacteria that are unable to activate the SOS response are highly sensitive to the antibiotics listed above and are less likely to develop resistance. The goal of this project is to discover drugs that prevent SOS activation and increase the efficacy of several important antibiotics.